Protein Science, Vol 3, Issue 8 1206-1212, Copyright © 1994 by Cold Spring Harbor Laboratory Press

ARTICLE

Properties of a recombinant human hemoglobin double mutant: Sickle hemoglobin with Leu-88({beta}) at the primary aggregation site substituted by Ala

JJM. DE-LLANO and J. M. MANNING
The Rockefeller University, New York, New York 10021

A recombinant double mutant of hemoglobin (Hb), E6V/L88A({beta}), was constructed to study the strength of the primary hydrophobic interaction in the gelation of sickle Hb, i.e., that between the mutant Val-6({beta}) of one tetramer and the hydrophobic region between Phe-85({beta}) and Leu-88({beta}) on an adjacent tetramer. Thus, a construct encoding the donor Val-6({beta}) of the expressed recombinant HbS and a second mutation encoding an Ala in place of Leu-88({beta}) was assembled. The doubly mutated {beta}-globin gene was expressed in yeast together with the normal human {alpha}-chain, which is on the same plasmid, to produce a soluble Hb tetramer. Characterizations of the Hb double mutant by mass spectrometry, by HPLC, and by peptide mapping of tryptic digests of the mutant {beta}-chain were consistent with the desired mutations. The absorption spectra in the visible and the ultraviolet regions were practically superimposable for the recombinant Hb and the natural Hb purified from human red cells. Circular dichroism studies on the overall structure of the recombinant Hb double mutant and the recombinant single mutant, HbS, showed that both were correctly folded. Functional studies on the recombinant double mutant indicated that it was fully cooperative. However, its gelation concentration was significantly higher than that of either recombinant or natural sickle Hb, indicating that the strength of the interaction in this important donor-acceptor region in sickle Hb was considerably reduced even with such a conservative hydrophobic mutation.
CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				Z. Liu, W. Weng, R. M. Bookchin, V. L. Lew, and F. A. Ferrone Free Energy of Sickle Hemoglobin Polymerization: A Scaled-Particle Treatment for Use with Dextran as a Crowding Agent Biophys. J., May 1, 2008; 94(9): 3629 - 3634. [Abstract] [Full Text] [PDF]

				L. R. Manning, J. E. Russell, J. C. Padovan, B. T. Chait, A. Popowicz, R. S. Manning, and J. M. Manning Human embryonic, fetal, and adult hemoglobins have different subunit interface strengths. Correlation with lifespan in the red cell Protein Sci., August 1, 2007; 16(8): 1641 - 1658. [Abstract] [Full Text] [PDF]

				M. Ashiuchi, T. Yagami, R. J. Willey, J. C. Padovan, B. T. Chait, A. Popowicz, L. R. Manning, and J. M. Manning N-terminal acetylation and protonation of individual hemoglobin subunits: Position-dependent effects on tetramer strength and cooperativity Protein Sci., June 1, 2005; 14(6): 1458 - 1471. [Abstract] [Full Text] [PDF]

				X. Li, R. W. Briehl, R. M. Bookchin, R. Josephs, B. Wei, J. M. Manning, and F. A. Ferrone Sickle Hemoglobin Polymer Stability Probed by Triple and Quadruple Mutant Hybrids J. Biol. Chem., April 12, 2002; 277(16): 13479 - 13487. [Abstract] [Full Text] [PDF]

				T. Yagami, B. T. Ballard, J. C. Padovan, B. T. Chait, A. M. Popowicz, and J. M. Manning N-terminal contributions of the {gamma}-subunit of fetal hemoglobin to its tetramer strength: Remote effects at subunit contacts Protein Sci., January 1, 2002; 11(1): 27 - 35. [Abstract] [Full Text] [PDF]

				R. M. Bookchin, T. Balazs, Z. Wang, R. Josephs, and V. L. Lew Polymer Structure and Solubility of Deoxyhemoglobin S in the Presence of High Concentrations of Volume-excluding 70-kDa Dextran. EFFECTS OF NON-S HEMOGLOBINS AND INHIBITORS J. Biol. Chem., March 5, 1999; 274(10): 6689 - 6697. [Abstract] [Full Text] [PDF]

				A. Dumoulin, J. C. Padovan, L. R. Manning, A. Popowicz, R. M. Winslow, B. T. Chait, and J. M. Manning The N-terminal Sequence Affects Distant Helix Interactions in Hemoglobin. IMPLICATIONS FOR MUTANT PROTEINS FROM STUDIES ON RECOMBINANT HEMOGLOBIN FELIX J. Biol. Chem., December 25, 1998; 273(52): 35032 - 35038. [Abstract] [Full Text] [PDF]

				A. Dumoulin, L. R. Manning, W. T. Jenkins, R. M. Winslow, and J. M. Manning Exchange of Subunit Interfaces between Recombinant Adult and Fetal Hemoglobins. EVIDENCE FOR A FUNCTIONAL INTER-RELATIONSHIP AMONG REGIONS OF THE TETRAMER J. Biol. Chem., December 12, 1997; 272(50): 31326 - 31332. [Abstract] [Full Text] [PDF]

				X. Li, U. A. Mirza, B. T. Chait, and J. M. Manning Systematic Enhancement of Polymerization of Recombinant Sickle Hemoglobin Mutants: Implications for Transgenic Mouse Model for Sickle Cell Anemia Blood, December 1, 1997; 90(11): 4620 - 4627. [Abstract] [Full Text] [PDF]

				J.-P. Himanen, A. M. Popowicz, and J. M. Manning Recombinant Sickle Hemoglobin Containing a Lysine Substitution at Asp-85(alpha ): Expression in Yeast, Functional Properties, and Participation in Gel Formation Blood, June 1, 1997; 89(11): 4196 - 4203. [Abstract] [Full Text] [PDF]

				J.-P. Himanen, U. A. Mirza, B. T. Chait, R. M. Bookchin, and J. M. Manning A Recombinant Sickle Hemoglobin Triple Mutant with Independent Inhibitory Effects on Polymerization J. Biol. Chem., October 11, 1996; 271(41): 25152 - 25156. [Abstract] [Full Text] [PDF]

				L. R. Reddy, K. S. Reddy, S. Surrey, and K. Adachi Role of Hydrophobic Amino Acids at beta 85 and beta 88 in Stabilizing F Helix Conformation of Hemoglobin S J. Biol. Chem., October 4, 1996; 271(40): 24564 - 24568. [Abstract] [Full Text] [PDF]

				J.-P. Himanen, K. Schneider, B. Chait, and J. M. Manning Participation and Strength of Interaction of Lysine 95([IMAGE]) in the Polymerization of Hemoglobin S as Determined by Its Site-directed Substitution by Isoleucine J. Biol. Chem., June 9, 1995; 270(23): 13885 - 13891. [Abstract] [Full Text] [PDF]